1. Field of the Invention
The present invention provides endotoxin-binding peptides which are useful in the treatment and prevention of sepsis. The present invention further provides a method for treating or preventing sepsis comprising administration of the compounds of the present invention.
2. Description of the Related Art
The incidence of sepsis in the United States is approximately 500,000 cases per year, of which the mortality incidence ranges from 35 to 65% (Dellinger, Opal et al. 1997). Despite the most modern of intensive life support measures, this means that 100,000 to 300,000 people die each year in the United States from endotoxic shock (sepsis). It is estimated that the annual cost in treating these patients is 5 to 10 billion dollars (Dellinger, Opal et al. 1997). Severe sepsis is initiated by an acute infection which leads to local and systemic release of bacterial products.
There are numerous, well characterized bacterial and host mediators of inflammation, all of which likely make a significant contribution in cases of overwhelming infection. It is also well known that the host inflammatory response leads to tissue injury, shock, and multiple organ dysfunction and in the majority of cases, release of bacterial endotoxins is the primary causative agent of endotoxic shock.
Endotoxin, or lipopolysaccharides (LPS) of gram-negative bacteria, are potent stimulators of the production and release of cytokines and pro-inflammatory mediators. Endotoxin causes activation of macrophages and induces the production of monokines, particularly tissue necrosis factor (TNF- and TNF-) and interleukin xe2x88x921 (IL-1) (Parrillo 1993; Tracey and Cerami 1993). Other mediators of inflammation include IL-6, IL-8, nitric oxide (NO) platelet activating factor, prostaglandin, leukotrienes, complement components, and kinins (Tracey and Cerami 1993). The cumulative effect of all these mediators is disseminated intravascular coagulation, multiple organ failure and most often, death.
Thus, development of an anti-LPS agent seems a rational approach to treatment of gram negative bacterial infection. However, whereas anti-TNF antibody treatment has proved somewhat effective in some selected clinical trials (Tracey and Cerami 1993; Tracey 1994), to date, targeting endotoxin with monoclonal antibodies as a method of treatment has proved ineffective. This may be because the antibodies lack in vivo neutralizing activity or because endotoxin is not the appropriate target for intervention. Or it may be because the antibodies do not possess sufficient cross-strain reactivities or because the antibodies do not bind LPS while it is still resident in the bacterial membrane. Regardless, there is a pressing demand for a clinically useful, safe therapy for removal of endotoxin from the bloodstream.
Different classes of anti-LPS agents are currently in development. Lipid A derivatives (such as E5331) may block or reverse endotoxin induced effects. Other agents, such as monophosphorylated Lipid A are meant to produce tolerance to endotoxin mediated effects and may have partial prophylactic utility. Still other agents in development include peptides, proteins, or lipoproteins which bind, neutralize and facilitate clearance of endotoxin. One such protein is recombinant heparin binding protein (rHBP; formerly known as cationic antimicrobial protein or bactericidal/permeability increasing factor) (Heinzelmann, Mercer-Jones et al. 2000). rHBP was administered to mice at different concentrations and at different intervals before and after cecal ligation and puncture. Survival was increased in mice pretreated well in advance with rHBP and was increased significantly, compared to controls, in animals treated with cefoxitin (an antibiotic) and rHBP. However, in animals treated with rHBP at the time of ligation and puncture, survival was not significantly different than that in the control group. Other proteins that have been tested include recombinant antithrombin III and LPS binding protein.
The interaction of LPS with its cognate binding protein has not yet been structurally elucidated. However, Ferguson et al (Ferguson, Welte et al. 2000) recently modeled the binding between LPS with the integral outer membrane protein FhuA from Escherichia coli. In accord with the results of previous mass spec and NMR studies, the model showed that the preponderance of stabilizing binding interactions occurred between 8 positively charged residues of FhuA (provided by Lys residue) and negatively charged functional groups resident on LPS. The three dimensional motif of FhuA was used to search the existing protein data base and a subset of 4 individual residues was found to be conserved in different proteins recognized to bind LPS. This subset of 4 residues was thus postulated to provide the structural pattern of recognition for binding LPS. Interestingly, LPS binding protein which binds circulating LPS or LPS embedded in whole bacteria also retains this conserved motif. Transfer of LPS from LPS binding protein to soluble or membrane bound CD14 receptor is thought to trigger the LPS signaling cascade which in turn, sets off the systemic inflammatory response. Ferguson et al. suggested that the conserved motif could serve as a template for molecular modeling of an LPS scavenger model which would be designed to reduced septic shock.
In the absence of an effective anti-LPS agent, other treatment paradigms are being tested. For instance, continuous venovenous hemofiltration has been used effectively in canine or porcine models of septic shock and in human intervention to remove inflammatory mediators from the circulation (Hoffinann, Hartl et al. 1996; Murphy, Fessler et al. 1997). Here, the blood flow is directed through a high flow ultrafiltration device which allows the relatively low molecular weight inflammatory mediators to be removed. The blood is then redirected into the patient. In the animal models, infusion of LPS causes severe hypodynamic circulatory state with significant depression of mean arterial pressure and cardiac output with a concomitant increase in pulmonary artery pressure. By and large, however, continuous ultrafiltration caused an improvement in global hemodynamics, increased hepatic blood flow, decreased blood lactate and nitrite/nitrate concentrations while also eliminating several anaphylatoxins from the circulation. High flow ultrafiltration is still under consideration as a treatment for sepsis.
Previously a family of multi-chain helix peptides have been developed which contain up to 4 copies of single helix sequences (see U.S. Pat. No. 5,877,133, the contents of which is incorporated herein by reference). These peptides present a conformer of high cationic charge density. The present inventors have unexpectedly discovered that three of these helix family members, namely Tris Arg Helix #3, Tetra Arg Helix #3 and Tris Arg Helix #3 (constrained) possess potent endotoxin binding activity. In an in vitro assay format, as little as 1 mg/ml of any one of these peptides quantitatively binds upwards of 1 xcexcg/mL endotoxin in either water or plasma solution. The consequence of binding is that endotoxin is quantitatively sequestered from the plasma or aqueous solution. The key points of the present invention are thus:
1. The specified helix family peptides can be used to bind endotoxin in an in vitro assay format and can thus be used as part of a diagnostic for the presence of endotoxin in physiological fluids, including plasma, saliva, urine, or feces or in tissue samples.
2. The specified helix family peptides can be used to bind endotoxin in vivo and thus can be used as a singular or conjunctive therapy for removing endotoxin from the circulation.
3. The specified helix family peptides can be immobilized on surfaces to act as an affinity trap for endotoxin in ultrafiltration or dialysis-type treatments.
4. The specified helix family peptides can be used to bind and remove endotoxin from plasma fractionation products and virtually all other pharmaceutical preparations intended for human or veterinary use.
5. The specified helix family peptides can be used as a diagnostic tool to detect endotoxin in plasma fractionation products and virtually all other pharmaceutical preparations intended for human or veterinary use.
6. The specified helix family peptides serve as a model framework for endotoxin binding upon which new analogs are being designed through a process of molecular dynamics and binding simulations.